The invention relates generally to methods of detecting target analytes and, more specifically, the development of a platform that enables the efficient and simultaneous creation of multiple detection assays and the use of such assays to detect a wide variety of target analytes, including biomolecules.
All immunoassays, regardless of their format or usage, require a reliable and sensitive detection system. The most sensitive immunoassays are radioimmunoassays. However, the ELISA is the most widely used. ELISA assays rely on enzyme-catalyzed detection, and typically employ calorimetric substrates that result in color formation. The enhanced sensitivity enabled by the BALISA will greatly improve the quality of immunologically based tests. The sensitive detection afforded by the BALISA offers other advantages over existing technologies. If one chooses to trade off sensitivity for a shorter assay time period, use of ultra-sensitive detection technology enables faster measurement of analytes. Finally, ultra-sensitive detection technology enables one to further dilute difficult samples such as meat. This improved sensitivity is especially critical in the detection of biomolecules which may be present in low concentrations, such as prions or microorganisms that have a very slow generation time, such as Mycobacterium tuberculosis. 
The invention entails the use of any bacteriophage, modified to carry a reporter gene, to which any analyte-recognizing moiety is attached via the capsid. The modified bacteriophage is used to attach to the target analyte. This attachment is identified via bacteriophage amplification in a helper bacteria strain with subsequent production and detection of the reporter gene. Assays according to the present invention are able to detect rapidly and with high sensitivity a large variety of biomolecules, including bacteria, viruses, toxins, bioterrorist agents such as anthrax spores, and prions.
The advantages of this system over other systems are numerous. One important advantage is sensitivity. The detection aspect of this assay is based on phage replication and enzymatic cleavage of a substrate and thus has two built-in signal amplification steps. Many bacteriophages can bind to a single target analyte. Consequently, the sensitivity of this assay is superior to other methods because as few as 100-1000 phages would be able to produce a detectable signal. In practical terms, this means that the system should be able to directly detect 10-100 particles of the target analyte, and the actual detection number is expected to be lower than that, as more than one bacteriophage will bind to a single analyte particle.
In addition, the method described here, in which multiple assays can be produced from a single bacteriophage, is advantageous and cost effective because the assays can be produced based on a standardized platform, without the need to genetically and phenotypically completely characterize new bacteriophages for every new test.
The assay combines two proven methods, reporter bacteriophage technology, and Enzyme linked Immunosorbent Assay (ELISA) into one integrated method. The integrated technology, known as the Bacteriophage Linked Immunosorbent Assay (BALISA) harnesses the signal amplification produced by bacteriophage amplification, and enzymatic cleavage of a substrate to produce a very sensitive assay, capable of rapid detection of the target biomolecule. In one specific use, the BALISA test described herein could be used to detect foodborne pathogens. Foodborne illness accounts for seventy-five million illnesses in the United States each year. In another use, the BALISA could be used to detect biological and chemical select agents, capable to be used as biological weapons. These agents include toxins, bacteria, and viruses. The BALISA can be used directly in the field, thereby allowing fast, sensitive detection of pathogenic microorganisms and toxins.